Mecanismos de diferenciación celular en una de las células eucariotas más primitivas, el protozoario intestinal Giardia lamblia

Giardia lamblia es un protozoario que habita en el intestino de seres humanos y otros vertebrados. La forma vegetativa del parásito carece de organelas típicas de células eucariotas tales como mitocondrias, peroxisomas y compartimentos relacionados en el tráfico intracelular y secreción de proteínas como el aparato de Golgi y gránulos de secreción. Dentro del intestino algunos trofozoítos se transforman en quistes, la forma infectiva, que se liberan con las heces, responsables de la transmisión de la enfermedad. El enquistamiento se manifiesta como un proceso de adaptación celular a la falta de colesterol que ocurre en la parte inferior del intestino, aunque no se conocen los mecanismos de transducción de señales que llevan a la expresión de genes específicos. Este proyecto está dirigido a conocer los aspectos del proceso de enquistamiento de Giardia, como son a) mecanismos de transducción de señales que se generan ante esta ausencia de colesterol y la regulación de la expresión de genes específicos, b) transporte intracelular de los componentes de la pared del quiste, en particular la biogénesis de las vesículas especificas de secreción y del aparato de Golgi, organelas presentes en trofozoítos en proceso de enquistamiento y c) el ensamblado de la pared extracelular.Fil: Luján, Hugo Daniel. Universidad Católica de Córdoba. Facultad de Ciencias de la Salud; ArgentinaFil: Carranza, Pedro Gabriel. Universidad Católica de Córdoba. Facultad de Ciencias de la Salud; ArgentinaFil: Saura, Alicia. Universidad Católica de Córdoba. Facultad de Ciencias de la Salud; Argentin

Mecanismos de diferenciación celular en una de las células eucariotas más primitivas, el protozoario intestinal Giardia lamblia

Giardia lamblia es un protozoario que habita en el intestino de seres humanos y otros vertebrados. La forma vegetativa del parásito carece de organelas típicas de células eucariotas tales como mitocondrias, peroxisomas y compartimentos relacionados en el tráfico intracelular y secreción de proteínas como el aparato de Golgi y gránulos de secreción. Dentro del intestino algunos trofozoítos se transforman en quistes, la forma infectiva, que se liberan con las heces, responsables de la transmisión de la enfermedad. El enquistamiento se manifiesta como un proceso de adaptación celular a la falta de colesterol que ocurre en la parte inferior del intestino, aunque no se conocen los mecanismos de transducción de señales que llevan a la expresión de genes específicos. Este proyecto está dirigido a conocer los aspectos del proceso de enquistamiento de Giardia, como son a) mecanismos de transducción de señales que se generan ante esta ausencia de colesterol y la regulación de la expresión de genes específicos, b) transporte intracelular de los componentes de la pared del quiste, en particular la biogénesis de las vesículas especificas de secreción y del aparato de Golgi, organelas presentes en trofozoítos en proceso de enquistamiento y c) el ensamblado de la pared extracelular.Fil: Luján, Hugo Daniel. Universidad Católica de Córdoba. Facultad de Ciencias de la Salud; ArgentinaFil: Carranza, Pedro Gabriel. Universidad Católica de Córdoba. Facultad de Ciencias de la Salud; ArgentinaFil: Saura, Alicia. Universidad Católica de Córdoba. Facultad de Ciencias de la Salud; Argentin

The giardial VPS35 retromer subunit is necessary for multimeric complex assembly and interaction with the Vacuolar protein sorting receptor

The retromer is a pentameric protein complex that mediates the retrograde transport of acid hydrolase receptors between endosomes and the trans-Golgi network and is conserved across all eukaryotes. Unlike other eukaryotes, the endomembrane system of Giardia trophozoite is simple and is composed only of the endoplasmic reticulum and peripheral vesicles (PVs), which may represent an ancient organellar system converging compartments such as early and late endosomes and lysosomes. Sorting and trafficking of membrane proteins and soluble hydrolases from the endoplasmic reticulum to the PVs have been described as specific and conserved but whether the giardial retromer participates in receptor recycling remains elusive. Homologs of the retromer Vacuolar Protein Sorting (Vps35p, Vps26p, and Vps29p) have been identified in this parasite. Cloning the GlVPS35 subunit and antisera production enabled the localization of this protein in the PVs as well as in the cytosol. Tagged expression of the subunits was used to demonstrate their association with membranes, and immunofluorescence confocal laser scanning revealed high degrees of colabeling between the retromer subunits and also with the endoplasmic reticulum and PV compartment markers. Protein-protein interaction data revealed interaction between the subunits of GlVPS35 and the cytosolic domain of the hydrolase receptor GlVps. Altogether our data provide original information on the molecular interactions that mediate assembly of the cargo-selective retromer subcomplex and its involvement in the recycling of the acid hydrolase receptor in this parasite.Fil: Miras, Silvana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Instituto de Investigaciones Médicas Mercedes y Martín Ferreyra; ArgentinaFil: Merino, Maria Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Instituto de Investigaciones Médicas Mercedes y Martín Ferreyra; ArgentinaFil: Gottig Schor, Natalia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Ropolo, Andrea Silvana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Instituto de Investigaciones Médicas Mercedes y Martín Ferreyra; ArgentinaFil: Touz, Maria Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Instituto de Investigaciones Médicas Mercedes y Martín Ferreyra; Argentin

Vacuolar protein sorting receptor in Giardia lamblia.

In Giardia, lysosome-like peripheral vacuoles (PVs) need to specifically coordinate their endosomal and lysosomal functions to be able to successfully perform endocytosis, protein degradation and protein delivery, but how cargo, ligands and molecular components generate specific routes to the PVs remains poorly understood. Recently, we found that delivering membrane Cathepsin C and the soluble acid phosphatase (AcPh) to the PVs is adaptin (AP1)-dependent. However, the receptor that links AcPh and AP1 was never described. We have studied protein-binding to AcPh by using H6-tagged AcPh, and found that a membrane protein interacted with AcPh. This protein, named GlVps (for Giardia lamblia Vacuolar protein sorting), mainly localized to the ER-nuclear envelope and in some PVs, probably functioning as the sorting receptor for AcPh. The tyrosine-binding motif found in the C-terminal cytoplasmic tail domain of GlVps was essential for its exit from the endoplasmic reticulum and transport to the vacuoles, with this motif being necessary for the interaction with the medium subunit of AP1. Thus, the mechanism by which soluble proteins, such as AcPh, reach the peripheral vacuoles in Giardia appears to be very similar to the mechanism of lysosomal protein-sorting in more evolved eukaryotic cells

Vacuolar Protein Sorting Receptor in Giardia lamblia

In Giardia, lysosome-like peripheral vacuoles (PVs) need to specifically coordinate their endosomal and lysosomal functions to be able to successfully perform endocytosis, protein degradation and protein delivery, but how cargo, ligands and molecular components generate specific routes to the PVs remains poorly understood. Recently, we found that delivering membrane Cathepsin C and the soluble acid phosphatase (AcPh) to the PVs is adaptin (AP1)-dependent. However, the receptor that links AcPh and AP1 was never described. We have studied protein-binding to AcPh by using H6-tagged AcPh, and found that a membrane protein interacted with AcPh. This protein, named GlVps (for Giardia lamblia Vacuolar protein sorting), mainly localized to the ER-nuclear envelope and in some PVs, probably functioning as the sorting receptor for AcPh. The tyrosine-binding motif found in the C-terminal cytoplasmic tail domain of GlVps was essential for its exit from the endoplasmic reticulum and transport to the vacuoles, with this motif being necessary for the interaction with the medium subunit of AP1. Thus, the mechanism by which soluble proteins, such as AcPh, reach the peripheral vacuoles in Giardia appears to be very similar to the mechanism of lysosomal protein-sorting in more evolved eukaryotic cells

AcPh localization and activity.

<p>(A) Schematic representation of the <i>acph</i> gene containing the GGTAAGCCTATCCCTAACCCTCTCCTCGGTCTCGATTCTACGCGTA. CCGGT and CATCATCATCATCATCAT, coding to the V5 epitope and six histidine residues, respectively. A 3D reconstruction of the gene product tagged with V5-H6 using the hidden Markov models (HMMs) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043712#pone.0043712-Soding1" target="_blank">[84]</a> and MODELLER <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043712#pone.0043712-Sali1" target="_blank">[85]</a> is also represented. (B) IFA and confocal microscopy show AcPh-V5 predominantly in the ER but also in the nuclear envelope and PVs (arrowheads). DIC: Differential interference contrast microscopy. (C) Acid phosphatase activity on the PVs and bare zone is observed by using the specific substrate ELF97 at pH 5.5. Alkaline phosphatase activity was not detected in trophozoites at pH ≥7.0. Nuclear DNA was labeled with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Bar, 10 μm.</p

GlVps and the medium subunit of AP1 interact via the YQII motif.

<p>(A) Densitometric assessment of one representative RT–PCR experiment shown on bottom. The amount of 1000 nt antisense RNA from the vector is only observed in −µ1 trophozoites. Reduction of endogenous μ1 mRNA levels is observed in −µ1, but not in +µ1 or wild-type cells (wt). Similar expression of <i>glvps</i> mRNA in wild-type, +µ1 and −µ1 cells was observed. (*p<0,0001). (B) GlVps-HA is observed in the cytoplasm in µ1-depleted cells. In cell expressing µ1 (+µ1), GlVps-HA possesses a reticular-perinuclear distribution. Merge panels of green fluorescence and differential interference contrast microscopy for +µ1 and −µ1 trophozoites are shown. Bar, 10 μm. (C) GlVps-HA is detected by immunoblotting using anti-HA mAb in +µ1 (a) and −µ1 (b) trophozoites. The proteolytic processing of GlVps-HA observed in −µ1 trophozoites, differs from the processing of GlVPS_-YQII-HA in cells expressing µ1 (c). Relative molecular weights of protein standards (kDa) are indicated on the left. (D) The yeast two-hybrid assay demonstrates that GlVps (GlVps-AD) but not GlVps_-YQII (GlVps-AD lacking the lysosomal motif) interacts with μ1 (μ1-BD) (left panel). GlVps (GlVps-AD) does not interact with the μ2 subunit of AP2 (μ2-BD) (right panel). Interaction is noticed by the growth of yeast colonies in plates lacking tryptophan, leucine and histidine [TDO (triple-dropout medium) plates] and in the high-stringency medium that also lacked adenine (QDO). Controls of the methodology include testing of pESCP-AD/pµ1-BD or pGlLRP-AD/pµ2-BD (protein-protein interaction) and pGlVps-AD/pGBKT7 (autoactivation).</p

The YQII motif of GlVps contributes to receptor stabilization.

<p>(A) GlVps_-YQII–HA (green) is observed in the cytosol (cyt) of trophozoites probable in small vesicles (white arrows in insert) by IFA and confocal microscopy. pm: plasma membrane. (B) GlVps_-YQII–HA (green) do not colocalizes with BiP (red) in the ER (Merge). Inset (a) magnifies a region of the cell and shows that the green and red fluorescence are well separated. Differential interference contrast microscopy (b) is shown as insert. Scatter plot (panel on the left) correspond to the colocalization analysis. (C) Partial colocalization of GlVps_-YQII–HA (green) and μ2 (red) is observed in the PV region (Merge). Inset (a) magnifies a region of the cell where the green and red fluorescence partially overlap in the PVs. Differential interference contrast microscopy (b) is shown as insert. Bars, 10 μm. Scatter plot of the two labels shows the colocalization (left panel). Pearson's coefficient (PC). Manders' Overlap coefficient (M). (D) GlVps-HA and GlVps_-YQII–HA are detected by immunoblotting using anti-HA mAb in <i>GlVps-ha</i>, <i>GlVps_-YQII–ha</i> trophozoites, respectively. No detection of these receptors was observed in wild-type cells. Proteolytic processing is observed for GlVps_-YQII–HA in comparison with GlVps–HA. Relative molecular weights of protein standards (kDa) are indicated on the left.</p

Expression of GL28954.

<p>(A) RT-PCR experiment show that the mRNA of GL28954 is expressed as predicted by the GiardiaDB. <b>1</b>: fragment of 1490 bp amplified using the primer pair F2/R1; <b>2</b>: the predicted 1653 bp ORF amplified using the primer pair F1/R1; <b>3</b>: expression of a <i>gdh</i> mRNA fragment was tested as positive control; <b>4</b>: DNA-contamination control. (B) Immunoblotting using anti-HA mAb shows the predicted band of 60 kDa for GL28954 (black arrow) but also a higher 120 kDa band that might correspond to GL28954 homodimer (gray arrow) in transgenic trophozoites (TT). Wild-type trophozoites (WT) do not show the presence of GL28954-HA. Relative molecular weights of protein standards (kDa) are indicated on the left. (C) IFA and confocal microscopy show the HA-tagged GL28954 mainly around the nuclei. DIC: Differential interference contrast microscopy. Nuclear DNA was labeled with DAPI (blue). Bar, 10 μm.</p

GlVps and AcPh colocalized throughout the lysosomal pathway.

<p>(A) Direct IFA and confocal microscopy show the colocalization (Merge) of GlVps (green) and AcPh-V5H₆ (red) using directed labeled anti-HA and anti-V5, respectively. Inset magnifies a region of the cell and shows colocalization of the green and red fluorescence in yellow (a). Differential interference contrast microscopy (b) is shown as insert. Scatter plot of the two labels confirms the colocalization (right panel). Bar, 10 μm. (B) AcPh/GlVps and AcPh/GlVps_-YQII interaction was detected by the ability of yeast cells (AH109) to grow on selective plates TDO. No interaction was observed in the high-stringency QDO medium. Controls of the methodology include testing of pESCP-AD/pµ1-BD (protein-protein interaction) and pGlVps-AD/pGBKT7 (autoactivation).</p